School of Marine Sciences, Sun Yat-sen University, Guangzhou, 510000, China; Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai, 519000, China; Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, Guangzhou, 510000, China.
Fish Shellfish Immunol. 2024 Mar;146:109408. doi: 10.1016/j.fsi.2024.109408. Epub 2024 Feb 1.
Small ubiquitin-like modifier (SUMO) is a reversible post-translational modification that regulates various biological processes in eukaryotes. Ubiquitin-conjugating enzyme 9 (UBC9) is the sole E2-conjugating enzyme responsible for SUMOylation and plays an important role in essential cellular functions. Here, we cloned the UBC9 gene from sea perch (Lateolabrax japonicus) (LjUBC9) and investigated its role in regulating the IFN response during red-spotted grouper nervous necrosis virus (RGNNV) infection. The LjUBC9 gene consisted of 477 base pairs and encoded a polypeptide of 158 amino acids with an active site cysteine residue and a UBCc domain. Phylogenetic analysis showed that LjUBC9 shared the closest evolutionary relationship with UBC9 from Paralichthys olivaceus. Tissue expression profile analysis demonstrated that LjUBC9 was significantly increased in multiple tissues of sea perch following RGNNV infection. Further experiments showed that overexpression of LjUBC9 significantly increased the mRNA and protein levels of RGNNV capsid protein in LJB cells infected with RGNNV, nevertheless knockdown of LjUBC9 had the opposite effect, suggesting that LjUBC9 exerted a pro-viral effect during RGNNV infection. More importantly, we found that the 93rd cysteine is crucial for its pro-viral effect. Additionally, dual luciferase assays revealed that LjUBC9 prominently attenuated the promoter activities of sea perch type Ⅰ interferon (IFN) in RGNNV-infected cells, and overexpression of LjUBC9 markedly suppressed the transcription of key genes associated with RLRs-IFN pathway. In summary, these findings elucidate that LjUBC9 impairs the RLRs-IFN response, resulting in enhanced RGNNV infection.
小泛素样修饰物（SUMO）是一种可逆的翻译后修饰，调节真核生物中的各种生物过程。泛素结合酶 9（UBC9）是唯一负责 SUMO 化的 E2 结合酶，在重要的细胞功能中发挥重要作用。在这里，我们从鲈鱼（Lateolabrax japonicus）（LjUBC9）中克隆了 UBC9 基因，并研究了其在调控红鳍东方鲀神经坏死病毒（RGNNV）感染期间 IFN 反应中的作用。LjUBC9 基因由 477 个碱基对组成，编码一个由 158 个氨基酸组成的多肽，具有活性位点半胱氨酸残基和 UBCc 结构域。系统进化分析表明，LjUBC9 与牙鲆的 UBC9 具有最密切的进化关系。组织表达谱分析表明，RGNNV 感染后鲈鱼的多个组织中 LjUBC9 显著增加。进一步的实验表明，在感染 RGNNV 的 LJB 细胞中，LjUBC9 的过表达显著增加了 RGNNV 衣壳蛋白的 mRNA 和蛋白水平，而 LjUBC9 的敲低则产生相反的效果，表明 LjUBC9 在 RGNNV 感染过程中发挥了促病毒作用。更重要的是，我们发现第 93 位半胱氨酸对其促病毒作用至关重要。此外，双荧光素酶报告基因实验显示，LjUBC9 显著减弱了 RGNNV 感染细胞中鲈鱼Ⅰ型干扰素（IFN）启动子的活性，而过表达 LjUBC9 则显著抑制了与 RLRs-IFN 通路相关的关键基因的转录。综上所述，这些发现表明 LjUBC9 削弱了 RLRs-IFN 反应，导致 RGNNV 感染增强。
